Voltage dependent anion channels (VDACs) are small outer mitochondrial membrane proteins found in all eucaryotes. VDACs are found in close association with the ADP translocator channel, and directly bind several kinases that exist both in a soluble cytosolic form and a membrane bound form. This group of kinases is important in a variety of metabolic functions, including glycolysis, triglyceride metabolism, insulin release, and insulin response, in addition to other metabolic pathways. The kinases known to bind VDACs include hexokinase, glucokinase, glycerol kinase, and mitochondrial creatine kinase. Binding to VDACs is metabolically and developmentally regulated in a tissue-specific fashion, and is enhanced in transformed cell lines. It has been reported that VDACs are also found in other membranes and are known to be a component of the peripheral benzodiazepine receptor, although the role of VDACs in this regard is unknown. While the isolation of the two human VDAC genes was recently reported, little is known about the physiologic function of VDACs in mammals. It would be very useful to identify VDAC genes from a mammal more amenable to experimental manipulation, to test the hypothesis that VDACs participate in a variety of metabolic pathways through binding of metabolically important kinases, and in mitochondrial respiratory function via regulated ADP transport. As a preliminary step in addressing these questions we have isolated three distinct VDAC genes. The goals of this project are to create mutations in the mouse VDAC genes through targeted disruption in embryonic stem cells. In addition, the specificity of each isoform for a particular kinase will be studied in a yeast expression system. The objective of targeted gene disruption is to evaluate the relative importance of VDACs in energy metabolism. Mitochondrial respiratory function and electrophysiological properties will be studied in cell lines harboring VDAC mutations, and mutant mouse strains, if viable, will be used to determine the relative importance of VDAC function in vivo. In particular glucose homeostasis and muscle energy metabolism will be examined to determine whether or not there is redundancy in VDAC function due to the presence of multiple isoforms.